Studies on a Defective Variant of Simian Virus 40 That Is Substituted ...

THE JOURNAL OP BIOLOGICAL CHEMISTRY Vol 252, No. 14, Issue of July 25, pp. 5124-5134, 1977

Prmted in U S.A.

Studies on a Defective Variant of Simian Virus 40 That Is Substituted with DNA Sequences Derived from Monkey II. STRUCTURE OF DNA

(Received for publication, February 7, 1977)

G. R. KOTESWARA RAO* AND MAXINE F. SINGER*

From the Laboratory of Biochemistry, National Cancer Institute, National Znstitutes of Health, Bethesda, Maryland 20014

The structure of a substituted, reiterated defective variant of the simian virus 40 genome has been analyzed. The DNA of the defective variant is a closed circular duplex resistant to restriction endonuclease R.EcoRI and slightly shorter than the genome of wild type simian virus 40. Analysis of the double-stranded DNA segments produced by the action of a variety of restriction endonucleases on the defective genome allow description of the molecule as follows. (a) The full DNA molecule contains four tandem repeats of a DNA segment containing both SV40 and monkey DNA sequences. (b) Three out of the four segments are identical and are about 23% of the wild type genome in length: one segment of the four is larger and contains an additional piece of DNA about 4.3% of a wild type genome in length, but is otherwise identical with the other three segments. (c) Portions of the defective variant that contain monkey DNA can be isolated as discrete segments by restriction endonuclease digestion. (d) Some of the sequences originating from monkey DNA are derived from the highly reiterated class of monkey DNA sequences; others may represent sequences derived from infrequently reiterated or single copy monkey sequences.

Upon serial undiluted passage of plaque-purified, wild type SV401 in permissive monkey kidney cells, a variety of defective variants accumulate. The genomes of the defective forms have been shown to contain deletions of wild type DNA sequences, repetitions and inversions of wild type sequences, and substitutions with sequences derived from the monkey

* Present address, Department of Biochemistry, Institute of Medi- cal Sciences, Banares Hindu University, Varanasi 221005, India.

$ To whom requests for reprints should be sent. 1 The abbreviations used are: SV40, simian virus 40; DNA I, closed

circular duplex DNA; DNA III, linear duplex DNA; (EcoRI res) DNA I, closed circular duplex DNA containing no sites sensitive to cleavage by restriction endonuclease EcoRI; restriction endonucleases are named according to the proposals of Smith and Nathans Cl), thus, the mixture of enzymes obtained from Haemophilus influenzas Rd is called endonuclease R. (HindIIIHindIII); fragments of DNA generated by restriction endonuclease cleavage are also named according to the proposals of Smith and Nathans (11, thus, the largest fragment generated by endonuclease R.HindIII cleavage of a given DNA molecule is called (HindIID-A, the next largest is called (HindIIIf-B, and so forth, cRNA is an RNA copy of a DNA molecule that is synthesized with Escherichia coli RNA-polymerase.

DNA. The relevant literature is reviewed in the accompanying paper (2). In that paper we report the isolation of an SV40 defective variant that is substituted with monkey sequences, is slightly shorter than the wild type genome, and is resistant to endonuclease R.EcoRI, an enzyme that cleaves wild type SV40 DNA at one site. Here we report experiments in which several restriction endonucleases were used to analyze the structure of the substituted defective genome. A map of the DNA, constructed on the basis of the fragments obtained upon digestion, is presented. DNA-DNA hybridization experiments with the fragments permitted placement of the sequences derived from monkey DNA on the map as well as an indication of what portions of the wild type SV40 genome are present in the defective. Similar analyses of other substituted defective variants of SV40, separately isolated, have previously been presented by other investigators (3, 4L2

EXPERIMENTAL PROCEDURES

Unless indicated all materials and procedures are as described in the accompanying paper (2).

Materials - The substituted defective variant of the SV40 genome that is termed CVP8/1IP2(EcoRI res) DNA I is described in the accompanying paper and was prepared as described in detail therein. The preparation and characterization of the 32P-labeled cRNA of the (HindII/HindIII)-E fragment of CVP8IlIP2(EcoRI res) DNA I has been described previously (5). We are grateful to Drs. S. Segal and M. Rosenberg of the National Cancer Institute for making the cRNA available to us. We are also grateful to them for the preparation of fragments from CVP8IlIP2(EcoRI res) DNA I that were labeled at the 5’ termini generated by either endonuclease R.HindIII or R.HindII cleavage. The preparation of these fragments will be described in detail elsewhere.3 Briefly, CVP8IlIPZ(EcoRI res) DNA I was digested to completion with endonuclease R.HindIII, or with endonuclease R.HincII and the digests were treated with alkaline phosphatase to remove the terminal phosphomonoester groups and then treated with [w~*P]ATP in the presence of polynucleotide ki- nase. The mixture of 32P-labeled fragments produced by endonuclease R.HindIII digestion ias then digested with endonuclease R.HincII, and the four resulting labeled fragments were separated by electrophoresis on polyacrylamide gels and eluted from the gels. A portion of the mixture of 32P-labeled fragments produced by endonuclease R.HincII digestion were separated by electrophoresis and the (HincII)-D fragment was eluted from the gels.

All wild type SV40 DNA was derived from plaque CVB, strain 777

2 M. Oren, S. Lavi, and E. Winocour, unpublished experiments. 3 M. Rosenberg, S. Segal, E. Kuff, and M. F. Singer (1977) CeZE,

in press.

5124

by guest on March 31, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Structure of a Host-substituted SV40 Variant 5125

virus (21, which was the plaque used to initiate the high multiplicity passages giving rise to defective CVP8/1/P2.

‘Glabeled DNA III was prepared from wild type SV40 DNA I by treatment with endonuclease R.EcoRI as described (2).

Digestion of Purified CVPBIl IP2(EcoRI resl DNA with Restriction Endonucleases -Endonuclease R.Hop II (61, an isoschizomer of endonuclease R.HpaII (71, was a gift from Dr. Roberto DiLauro of the National Cancer Institute and was also prepared in this laboratory according to Takanami (6). Digestions were carried out with an excess of enzyme in 6 mM Tris.HCl, pH 7.9, 6 mM MgCl,, 6 rnM Z- mercaptoethanol at 37” for 2 h. Reactions were stopped by addition of EDTA to 0.05 M. For preparative purposes, digests were deprotein- ized with chloroform:isoamyl alcohol (24:1, v/v) and the solution was then concentrated by lyophilization, electrophoresed on 1.4% cylindrical agarose gels, and DNA fragments recovered as described (2).

Endonuclease R.Bam I (8) was obtained from Bethesda Research Laboratories, Inc; endonuclease R-Hind111 (9-11) from Miles Chemi- cal Co; endonuclease R.HincII (121, an isoschizomer of endonuclease R-Hind11 (71, from New England Biolabs; digestion conditions for all three enzymes were the same as described for endonuclease R.Hop II.

Gel Electrophoresis - Agarose (1.4%) and polyacrylamide gel electrophoresis (4% acrylamide, acrylamide:bisacrylamide, 20/l) was carried out both on slabs and in cylinders (6 mm diameter) under the conditions described previously (21. The polyacrylamide slab gels were run for 15 min at 50 V prior to addition of samples (in 6% glycerol, 0.25% sodium dodecyl sulfate, 0.1% bromphenol blue); electrophoresis was for 2 to 3 h at SO V (lo-cm-long gel) or 130 V (15-cm- long gel) at room temperature. Agarose slab gels were run for 3 to 5 h at 50 V. Slab gels were stained with ethidium bromide and visualized and photographed with short wave ultraviolet light (71, or radioauto- graphed.

Estimation of Size of Restriction Fragments - The size of the double-stranded DNA fragments produced by restriction endonuclease cleavage of DNA was estimated from the mobilities of the fragments on polyacrylamide gel electrophoresis compared to the mobilities of marker fragments. The fragments produced by cleavage of wild type SV40 DNA, strain 777, with either the mixture of endonucleases R-Hind11 and III or endonuclease R.HindIII alone served as markers. The sizes of the marker fragments have previously been reported (10) and procedures were as described by Danna et al. (IO). The size of all DNA fragments is expressed as per cent of a complete wild type SV40 genome. Double-stranded circular wild type SV40 DNA is approximately 3 x lo6 daltons which is equivalent to about 5000 base pairs (10).

Centrifugation - Procedures were as described (21. Neutral sucrose gradients (5 to 20%) were carried out in 0.1 M NaCl for 3 h in the SW50.1 rotor of the Spinco L5-65 centrifuge at 49,000 rpm and IO”.

Filter Hybridization - Hybridization of labeled DNA fragments (13-15) or 32P-labeled cRNA (5) to DNA immobilized on nitrocellulose filters was carried out in 50% formamide as previously described. DNA fragments were denatured, prior to hybridization, in 100% formamide for 1 h at 68”. Thereafter the samples were brought to the proper buffer conditions and 50% formamide by appropriate addi- tions and added to the filters. After 30 min to 1 hat 68”, hybridization was carried out for at least 24 h at 37” prior to washing of the filters.

DNA-DNA Hybridization in Solution-A detailed description of the experiment is given in the appropriate legend. The conditions used for reannealing of denatured DNA were those described by Lee et al. (31. At indicated intervals samples were removed into pre- chilled tubes and kept in ice until further processing. The extent of reannealing was followed by measuring the amount of DNA that became resistant to degradation by the single strand specific nuclease Sl (16). Sl nuclease was obtained from Miles Laboratories, Inc. Samples were adjusted to the following conditions by the addition of a concentrated mixture prior to treatment with Sl nuclease: 0.04 M sodium acetate, pH 4.4, 2 rnM ZnSO,, 15 @g/ml of heat- denatured calf thymus DNA. Sl was added at a concentration of 1 x lo3 units/ml. Incubation was for 20 min at 50” and the reaction was stopped by cooling in an ice bath and addition of carrier RNA to a final concentration of 20 kg/ml and trichloroacetic acid to a final concentration of 10%. The samples were filtered through nitrocellulose filters, the filters washed, dissolved in methyl cellosolve, and counted appropriately.

Preparation of 32P-labeled DNA by Nick Translation- DNA labeled with 32P was prepared by the combined action of DNase and Escherichia coti DNA polymerase I in the presence of [a-3ZP]dTTP, the other unlabeled deoxynucleoside triphosphates, and the unla-

beled DNA of interest (17,181. This is a procedure commonly referred to as “nick translation.” The procedure used in this laboratory was as described (191. We are grateful to Dr. M. Botchan of the Cold Spring Harbor Laboratory for a sample of 32P-labeled SV40 strain 777 DNA prepared by nick translation.

Direct Transfer of Separated DNA Fragments from Agarose Gels to Nitrocellulose Sheets - DNA fragments generated by cleavage with restriction endonucleases were separated by electrophoresis on agarose gels. The fragments were then transferred directly to sheets of nitrocellulose (Millipore Corp.) by the procedures described by Southern (20) using the modifications of Botchan et al. (21).

RESULTS

Table I lists the fragments of double-stranded DNA obtained by cleavage of CVP8Il/PZ(EcoRI res) DNA I with several restriction endonucleases as described in detail below. Fig. 1 summarizes the structural analysis and indicates the position of the various fragments within the larger, repeating segments that occur within the defective genome. We hope that Table I and Fig. 1 will assist the reader in following the detailed data.

Digestion of CVP8IIlP2(EcoRI res) DNA I with Endonucle- ase R .Hap II-Purified CVP8IlIP2(EcoRI resl DNA I was digested with endonuclease R.Hap II. This enzyme is known to make a single cleavage in wild type SV40 DNA I at 0.74 on the map (7, 22). Complete digestion of the defective genome, as monitored both by the disappearance of all the original DNA I, and by simultaneous experiments in which wild type DNA I was completely converted to full length DNA III yielded a single peak of DNA of about 9.7 S on neutral sucrose gradients (Fig. 2a). Analysis of the same endonuclease R,Hap II digest on 1.4% agarose gels indicated that the peak of material seen on sucrose gradients was in fact composed of two species of linear duplexes; the slower moving peak is called (Hap 111-A and the faster moving peak (Hap ID-B (Fig. 2b). Table II summarizes the data on the characterization of fragments (Hap 11)-A and (Hap ID-B. 3H-labeled (Hap ID-A and (Hap 111-B were eluted from the agarose gels and electrophoresed on polyacrylamide gels with markers of 14C-labeled endonuclease R~HindIIIHindIII fragments of wild type SV40 DNA (not shown). These cylindrical gels were sliced and counted and the

TABLE I

Summary of products of restriction endonuclease digestion of CVPBIl IP2 (EcoRI res) DNA I

The detailed description of the DNA fragments is presented in the text and figures and legends. The nomenclature is as described.’ Note that (Hind&C and (HindII/HindIII~-D are identical, as endonucleases R.HincII and R.HindII are isoschizomers (7).

Restriction endonuclease Size (Per cent of wild type SV40)

Hap II

Barn I

Hind111

HincII

HindII/HindIII

(Hap 111-A 26 (Hap ID-B 23 (Barn 11-A 22 (Barn 11-B 4.3 (HindIIIl-A 17 (HindIIIl-B 13 (HindIID-C 10 (HincID-A 17 @Yin&)-B 7.7 (HincID-C 4.5 (HindIl/HindIII)-A 8.9 (HindIIIHindIIIl-B 7.2 (HindII/HindIII)-C 4.7 (HindII/HindIII)-D 4.3 (HindIIIHindIIIl-E 3.1


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

5126 Structure of a Host-substituted SV40 Variant

HAP II HAP II

1 BIG’) 5 A

s

~, D,, C 2 E, BIE.1’

4.7 8.9 L

’ L

4.3 ’ 4.7 i 3.1’ HAP II A

3.1

HAP II HAP II I 1

EUC’I 2 A r , C 2 E , B(E’)

s L

4.7 8.9 ’

4.7 s 3.1 ’ HAP II 8

3.1

5 HINDIII ; ( HIND II ; > 8AM I

FIG. 1. Summary of restriction endonuclease fragments obtained from CVP8/1/P2(EcoRl res) DNA I and their relative positions. See text for explanation. The fragments lettered A, B, C, D, and E are those produced by digestion with endonucleases R. (HindII/HindIID. The two portions of (HindII/HindIII)-B indicated as C’ and E’ are produced by the cleavage of (HindII/HindIII)-B by endonuclease R.HopII. C’ and E’ are indistinguishable in size from (HindIIl HindIID-C and -E, respectively. The numbers refer to the size of each fragment expressed as per cent of the size of an SV40 wild type genome.

x f

10 20 30 40 50 60 70 80 90 loo

FRACTION NUMBER

FIG. 2. Digestion of CVPSIlIP2(EcoRI res) DNA I with endonuclease R.Hop II. 3H-labeled CVP8/1/P2(EcoRI res) DNA I (about 7500 cpm, 01 was treated with R.Hap II and W-labeled wild type SV40 DNA III (0) was added as a marker. a, one portion (20 ~1) was centrifuged on a 5 to 20% neutral sucrose gradient. Sedimentation was from right to left. 5, another 20-~1 portion was electrophoresed on a 1.4% cylindrical agarose gel at 60 V for 9 h. Movement was from left to right.

sizes of (Hap ID-A and (Hap 11)-B were calculated from their mobilities relative to the mobilities of the wild type fragments as described under “Experimental Procedures.” (Hap 111-A was about 26% of a wild type SV40 genome, and (Hap ID-B, 23% (Table II). These determinations are confirmed by the data shown in Fig. 3. Slot 6 shows the mobility of the two Hap II fragments compared to the mobility of wild type SV40 HindIIIHindIII fragments in Slots 1 and 7. (Hap 111-A is slightly larger than wild type (HindII/HindIII)-A fragment, and (Hap 111-B is slightly smaller than the wild type (Hind111 HindIII)-A fragment. The wild type (HindII/HindIII)-A is 22.5% of wild type genome in length (10). Similarly, length measurements of the two fragments on electron micrographs indicated that both were about 25% of wild type SV40 in length (Table II).

From the sizes of the two Hap II fragments and the relative numbers of counts in each fragment as determined on 1.4%

TABLE II

Properties of the (Hap II)-A and (Hap II)-B fragments obtained from CVPBIl lP2(EcoRI resi DNA I

The size of the fragments was estimated by polyacrylamide electrophoresis with markers of SV40 strain 777 (HindII/HindIII) fragments as described in the text. The sizes were also determined by contour length measurements on electron micrographs containing strain 777 DNA III as a length standard. The molar ratios of (Hap ID- A and (Hap ID-B were calculated from the sizes of the two fragments and the relative number of counts per min present in each fragment after separation on agarose gel electrophoresis as shown in Fig. 26. The data are the averages of three separate determinations. 3H- labeled flap ID-A and (Hap ID-B were hybridized to filters containing strain 777 SV40 DNA I and monkey DNA as described (21. The data on the molar ratios of the R. (HindII/HindIII) digestion products of (Hap ID-A and (Hap ID-B are derived from the experiment shown in Fig. 6 by the procedures described in Table III.

Fragment popery

Wap 111-A (Hap 11)-B

Size (per cent of SV401 by gel 26 23 by electron microscopy 25 25

Molar ratio 1 2.6 Per cent hybridized to filters with:

SV40 DNA 21 14 Monkey DNA 31 42

Molar ratio of R. (HindIIIHindIII) products

Fragment: A 0.9 1.0 B 0.0 0.0 C 1.7 1.4 D 0.5 0.0 E 2.4 2.6

agarose gels such as the one in Fig. 2b, the molar ratios of the two fragments were calculated (Table II). An average of three determinations gave 2.6 mol of (Hap ID-B to one of (Hap 111-A.

(Hap 11)-A and (Hap 111-B were tested for their ability to hybridize to wild type SV40 DNA and monkey DNA (from BSC-1 cells) on filters. The data are summarized in Table II. Both fragments hybridized to both SV40 DNA and monkey DNA, indicating the presence of SV40 sequences and of monkey repetitive sequences.

Digestion of CVP8Il IPXEcoRI resl DNA I and (Hap IO-A and (Hap II&B with Endonuclease R .Bam I- Endonuclease R.Bam I is known to make a single cleavage in wild type SV40 DNA at position 0.16 on the SV40 genome (23). Fig. 3, Slot 8 shows the results of digestion of CVPSIl/P2(EcoRI res) DNA I with endonuclease R.Bam I. A major band about the same size as fragment (Hap 11)-B was produced. The conversion of CVP8/1/P2(EcoRI res) DNA I to a fragment about 22 to 23% of a wild type genome in length was confirmed by neutral sucrose gradient centrifugation in the presence of a marker of wild type component III (data not shown). As pointed out in the legend to Fig. 3, the preparation of DNA used was probably contaminated with small amounts of wild type and CVPW P2(EcoRI sens) DNA I (2), thus giving rise to a few faint extra bands in the upper portion of the gel, including one the size of the expected wild type Bum I product, that is, DNA III (see Slot 9, for example). In addition, there is a faintly visible band, migrating between the wild type fragments J and K. (This band may not be visible upon reproduction.) The latter are 4.5 and 4.0% of a wild type genome in length, respectively (lo), and the relative mobility of the band generated from


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


FIG. 3. Digestion of CVP8Il/P2(EcoRl res) DNA I with various restriction endonucleases. In each case about 1 pg of DNA was treated with enzyme and the samples were then electrophoresed on a 4% polyacrylamide slab gel and the gel stained with ethidium bromide. The gel slots contained the following DNA samples. 1, SV40 strain 711 treated with R.(HindII/HindIII); 2, SV40, strain 771 treated with R.Bam I followed by treatment with R.(HindIIl HindIID; 3, CVP8IlIP2(EcoRI res) treated with R.Hap II and then R. (HindIIIHindIII); 4, CVP8/1/P2(EcoRI res) treated with R.Bam I and then R~(HindII/HindIII); 5, CVP8IlIP2(EcoRI res) treated with R. (HindIIIHindIII); 6, CVP8IlIP2(EcoRI res) treated with R.Hap II; 7, SV40 strain 777 treated with R~(HindII/HindIID; 8, CVP8/1/ P2(EcoRI res) treated with R.Bam I; 9, SV40 strain 777 treated with R.Bam I. The letters at the right indicate the positions of the fragments produced by action of R. (HindIIIHindIIIl on CVP8/1/ P2(EcoRI res) DNA I, as shown in Slot 5. Note that the preparation of CVP8IlIP2(EcoRI res) DNA I was contaminated with some other species, presumably CVP8/1/P2 (EcoRI sens) DNA, and wild type DNA (2).

CVP8/1/P2(EcoRI res) DNA I indicates that it is 4.3% of a wild type genome, in length. Thus, endonuclease R.Bam I cleavage of the substituted defective variant yields mainly irag- ments about 22% of the wild type in length ((Bum D-A), and a small amount of a fragment 4.3% in length ((Bum 11-B).

The results obtained when purified (Hap 111-A and (Hap II)- B are each treated with endonuclease R*Bam I are shown in Fig. 4, B and C. The main products are seen as rather broad bands on 4% polyacrylamide gels. The sizes can be estimated by comparing the mobilities of the products with the mobilities of wild type SV40 (HimlIIIHindIII) fragments run simultane-

‘ZLi,, - 10 20 30 40 50 60 70 80 93100110120130

FIG. 4. Digestion of fragments (Hap 11)-A and (Hap 111-B with endonuclease R.Bum I. 3H-labeled (Hap 111-A (4.7 x lo3 cpm) and (Hap ID-B (6.6 x lo3 cpm) were treated with R.Bum I and electrophoresed on 4% cylindrical polyacrylamide gels. One-millimeter slices were collected. (LO), 3H counts per min; (0- - -01, 32P counts per min. A, control of R. (HindII/HindIII) digest of 3ZP-labeled strain 777 SV40 DNA I mixed with 3H-labeled (Hap 111-A and a small amount of (Hap ID-B; B, R.Bam I digest of (Hap ID-A, C, R.Bam I digest of (Hap ID-B.

ously but on separate cylindrical gels (Fig. 4A). The fragments produced from both (Hap 111-A and (Hap 11)-B are between 11 and 13% of a wild type genome in size, that is, (Hap 11)-A and (Hap ID-B are cut approximately in half by endonuclease R-Barn I. A small amount of material, about 4% of a genome in length, is produced from (Hap ID-A (about Slice 115 in Fig. 4B), but not (Hap ID-B. The control in Fig. 4A shows (Hap II)- B and (Hap ID-A undigested with Bum I.

Thus, both endonuclease R*Hap II and endonuclease R.Bum I cleave CVPWlIP2(EcoRI res) DNA I into fragments about one-quarter the length of a wild type genome, with the Bum I cut being about midway between the Hap II cuts. Further, the slightly longer (Hap 11)-A appears to contain two Bum I sites, yielding both a 22% fragment and a 4% fragment, thus accounting for the 26% size of (Hap ID-A.

In Fig. 4, B and C some DNA appears to remain close to the top of the gel. This represents a problem with this gel, not incomplete digestion, since the undigested Hap II fragments migrated, as expected, just near wild type SV40 (HindIIl HindIII)-A fragment in mixed gels (Fig. 4A).

Digestion of CVP8IlIP2(EcoRI res) DNA I, and Its (Hap W-A, (Hap ZZ)-B, and (Bum 0-A Products with Endonuclea- ses R .HindlllHindZZZ- The live distinctive fragments that are produced upon total digestion of CVPWP2(EcoRI res) DNA I with the mixture of endonucleases R.HindII/HindIII are shown in the accompanying paper (Ref. 2, Fig. 5, a and b). They are called, in order of decreasing size, (HindII/HindIII)- A, -B, -C, -D, and-E. They can also be seen inSlot 5 of Fig. 3 of this paper. Fig. 5 shows a plot of the mobility of the wild type SV40 (HindIIIHindIII) fragments against the known size of those fragments (lo), expressed as per cent of a full wild type SV40 molecule. The mobilities of the fragments generated by endonucleases R~HindIIIHindIII from CVPWlIP2(EcoRI res) DNA I are indicated by arrows. The size of the live fragments generated from the defective as determined in Fig. 5 are


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Structure of a Host-substituted SV40 Variant

IL 25 50 75 100 125 150

SLICE NUMBER

FIG. 5. Size of (HindIIIHindIII) fragments obtained from CVPS/l/ P2(EcoRl res) DNA I. The data are from Fig. 56 of the accompanying paper (2). The procedures are described under “Experimental Proce- dures.” The size of the known wild type fragments is expressed as per cent of the complete wild type SV40 genome on the ordinate. The mobility is expressed as slice number on the abscissa. The mobilities of the (HindII/HindIII)-A, -B, -C, -D, and -E fragments of CVPS/l/ P2(EcoRI res) DNA I are indicated by the arrows.

shown in the first column of Table III. The second column gives the actual per cent of the total 3H radioactivity in each fragment, and the last column gives the calculated molar ratio of the various fragments. The low amount of the (HindIIl HindIII)-D fragment indicates that all the molecules in CVPEU l/P2(EcoRI res) DNA I do not yield this fragment.

(Hap II)-A and (Hap 11)-B were also digested with endonuclease R.HindII/HindIII and electrophoresed on cylindrical polyacrylamide gels with markers of the (HindII/HindIII) fragments generated from wild type SV40. The results are shown in Fig. 6. The digest of (Hap 11)-A (Fig. 6u) indicates the presence of the (HindII/HindIII)-A, -C, -D, and -E fragments that were identified in the digest of total CVP8/1/ P2(EcoRI res) DNA I. The original (HindII/HindIII)-B is missing, indicating that the endonuclease R.Hap II cleaved within the sequence of the (HindII/HindIII)-B fragment. How- ever, no new size fragment appears as a result of this cleavage and it therefore seems that endonuclease R. Hap II cut (HindII/HindIII)-B in such a way as to yield fragments the same size as two of the original (HirzdWHindIII) fragments. The absence of (HindII/HindIII)-B after endonuclease R.Hap II cleavage of CVP8/1/P2(EcoRI res) DNA I can also be seen by comparing Columns 3 and 5 in Fig. 3. The stoichiometry of the (HindIIIHindIII) fragments obtained from (Hap 11)-A is presented in Table II. The mole ratio of fragments the size of (HindII/HindIII)-C and -E is markedly increased over that seen for the same size fragments when the total DNA was digested with endonucleases R.HindII/HindIII (Table III), suggesting that (HindII/HindIII)-B fragment sequence was cleaved once by endonuclease R.Hap II thus yielding one fragment about 4.5% and one about 3% of a wild type genome in length (that is, the size of (HindII/HindIII)-C and -E, respectively) after digestion with both enzymes. (Hinda/ HindIII)-B was estimated to be 7.2% in length (Table III). Similar results were obtained upon digestion of (Hap 11)-B with endonucleases R.HindII/HindIII (Fig. 6bJ: the digest contained fragments corresponding to (HindII/HindIII)-A, -C, and -E. (HindII/HindIII)-B was missing, no new size classes of fragments were generated, and the yield of fragments the size of (HindII/HindIII)-C and -E (Table II) was increased com-

TABLE III

Endonuclease R. (HindIIlHindIII) fragments of CVPBIl lP2(EcoRI res) DNA I

The sizes of the fragments were determined as indicated in Fig. 5 and are expressed as percentages of the wild type SV40 genome. The per cent of the total counts per min in each fragment was determined from the data shown in Fig. 5 of the accompanying paper (2). The molar ratios of the fragments were obtained by dividing the per cent of the total counts per min in each fragment by the size of the fragment and normalizing the resulting ratios.

Fragment Size Total counts per min Molar ratio

% % A 8.9 43 1.3 B 7.2 25 1.0 c 4.7 11 0.6 D 4.3 2.8 0.2 E 3.1 10 0.9

GEL SLICE NUMBER

FIG. 6. Digestion of (Hap ID-A and (Hap ID-B with endonuclease R. (HindIIIHindIII). For this experiment 3H-labeled (Hap ID-A (4.3 x 10” cpm) and 3H-labeled (Hap 11)-B (6.1 x lo3 cpm), were each mixed with 13.6 x lo3 cpm of 32P-labeled strain 777 SV40 DNA and digested with endonuclease R. (HindII/HindIII). The reaction mix- tures were electrophoresed on 5% polyacrylamide gels (2) and l-mm slices were collected and counted: (0- - -O), 32P-labeled 777 SV40 DNA; C-01, 3H-labeled Hap II fragment. a, (Hap ID-A; b, (Hap ID-B.

pared to the yield from CVP8/l/P2 (EcoRI res) DNA I (Table III). Therefore, it appears that the two new fragments migrate with the original (HindII/HindIII)-C and -E.

The data in Fig. 6 also localize the CVP8/1/P2(EcoRI res) DNA (HindII/HindIII)-D fragment in the (Hap 11)-A fragment. (HindII/HindIII)-D appears in the digest of (Hap 11)-A (Fig. 6u), but not in the digest of (Hap 11)-B (Fig. 66) (summarized in the stoichiometries reported in Table II). The data are consistent with the following formulations which are summarized in Fig. 1. The two fragments, (Hap 11)-A and (Hap 11)-B are very similar, except that (Hap II)-A contains (HindIIl Hi&II)-D. Thus, (Rap II)-A is composed of one each of (HindII/HindIII)-A, -C, -D, and -E fragments, as well as one each of fragments we shall call C’ and E’ (see Fig. l), derived from the splitting of the original (HindII/HindIII)-B sequence by endonuclease R.Hap II. (Hap ID-B is identical except for the absence of the (HindII/HindIII)-D fragment. Adding up the sizes of the (HindIIIHindIII) fragments assigned to (Hap 11)-A, a molecule about 28% of the wild type in length is generated, while for (Hap ID-B a molecule about 24% in length is generated. These results are consistent with the estimated sizes of (Hap 11)-A and (Hap 11)-B discussed above


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


and with the stoichiometric relation between the fragments, considering the errors inherent in the various methods.

Column 4 of Fig. 3 shows that combined digestion of CVP8I lIP2(EcoRI res) DNA I with endonuclease R*Bam I and R. (HindIIIHindIII) gives all the expected (HindIIIHindIII) fragments, with the exception of fragment A. Thus, it appears that there is an endonuclease R.&m I cleavage site in (HindII/HindIII)-A. Since total CVP8/1/PZ(EcoRI res) DNA I was used for this experiment, the results again confirm the relatedness of the (Hap ID-A and (Hap 111-B fragments; the (HindIIlHindIID-A fragments of both (Hun ID-A and (Hap 11)-B are cleaved by endonuclease R.Bum I.

As seen in Fig. 3, Column 4, combined digestion of CVPS/lI P2(EcoRI res) DNA I with endonucleases R.Bam I and R. (HindIIIHindIII) did not yield any fragment of a new size class. We demonstrated above that cleavage of intact CVP8/1/ P2 (EcoRI res) DNA I with endonuclease R.Bum I converts the bulk of the DNA into fragments about 22% of the wild type genome in length and also yields a small fragment, about 4.3% in length. The most likely site for the Bum I cleavage in (HindII/HindIII)-A fragment is shown in Fig. 1. This cleavage would yield one fragment about 7.8% in length which would migrate with (HindII/HindIII)-B and another fragment, about 1.5% in length, which would not have been detected in our gels. Alternatively, the cleavage in (HindIIlHindIIIJ-A might have been such as to yield one fragment about the size of (HindII/HindIII)-C (about 4.1% and another about the size of (HindII/HindIII)-E (about 3.1%). The former interpretation is most consistent with the size of (HindII/HindIII)-A and is supported by the fact that endonuclease R.Bam I appears to cleave about midway between the endonuclease R*Hup II sites on the DNA. The structure for (Hap ID-A shown in Fig. 1 accounts for the formation of a 4.3% fragment by endonuclease R*Bum I and predicts that (HindIIlHindIID-D would be lost upon combined digestion with endonucleases R-Bum I and R. (HindIIIHindIII). Inspection of Fig. 3, Column 4, shows that the fragment is indeed missing.4

Digestion of CVP8Il lP2(EcoRI res) DNA I with Endonucle- uses R .HindZZZ and R .HincZZ- The experiment shown in Fig. 7 demonstrates that cleavage of the EcoRI-resistant defective variant with endonuclease R.HindIII alone produces three fragments, termed (HindIII)-A, -B, and -C. The sizes of the three fragments were calculated from gels such as those shown in Fig. 7 and these data as well as the stoichiometry of the fragments are summarized in Table IV. The three fragments are approximately 17, 13, and 10% of a full genome in size, respectively. In the experiment summarized in Table IV, the 3ZP-labeled CVP8/1/P2(EcoRI res) DNA I was obtained by nick translation of unlabeled DNA. From the size and the per cent of the total radioactivity in each fragment, the relative number of copies of each fragment was obtained and normalized (last column, Table IV) so that (HindIII)-A, which is present in the smallest amount, is taken as one. There are, for each copy of (HindUI)-A, about two of (HindID)-B and three of (HindIII)-C. This could be explained if there were 1 mol of a sequence yielding 1 mol each of (HindIII)-A and (HindIII)-C, and 2 mol of a sequence yielding (HindUI)-B and (HindUI)-C. As shown on Fig. 8, the (HindIII)-B fragment (SZots b and c) was cleaved by endonucleases R. (HindIIIHindIII) (that is, by endonuclease R. HindII) to give fragments the size of (Hi&II/

’ Recent unpublished experiments by H. Rosenberg and M. Rosen- berg with isolated (HindII/HindIII)-D confirm the presence of the endonuclease R.Bam I site.

FIG. 7. Digestion of CVPWlIP2(EcoRI resl DNA I with endonuclease R.HinldIII: determination of sizes of fragments on polyacrylamide gel electrophoresis. CVP8IlIP2(EcoRI resl DNA I was digested with endonuclease R.HindIII, electrophoresed on a 15-cm- long 4% polyacrylamide slab, and the gel stained with ethidium bromide. Slot a, markers of R. (HindIIIHindIII) digest of strain 777 SV40 DNA; only fragments A, B, C plus D, E, and F are visible; Slot b. markers of R. (HindIIIHindIIIl digest of CVPSIlIP2(EcoRI resl DNA I; only fragments A and B are clearly visible; SZot c, digest of CVP8/1/P2 (EcoRI resl DNA I with R.HindIII: the letters at the right correspond to the Hind111 fragments produced in Slot c.

HindUI)-A and-C (Slot d). Therefore, (HindIII)-B is composed of fragments (HindII/HindIII)-A and -C. This conclusion is incorporated into the diagram in Fig. 1. The size of (HindIn)- B, about 13% of the wild type genome, is consistent with this interpretation. Similarly, Fig. 8 shows that cleavage of the (HindIII)-C fragment (Fig. 8, e and f, with endonuclease R.HindII yielded fragments the size of (HindII/HindIII)-B and -E (Fig. f!g).

The following observations confirm the presence of the (HindII/HindIII)-E fragment in (HindIn)-C. As reported in the accompanying paper (2), CVP8/1/P2(EcoRI res) DNA I contains sequences homologous to repetitive DNA sequences in the host monkey cells and such sequences are present in


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


TABLE IV

Endonuchse R .HindIII fragments of CVPBIl /P2(EcoRI res) DNA I

The sizes of the fragments were estimated from the mobility of the HindIII fragments shown in Fig. 7 and similar experiments. The per cent of the total counts per min was estimated as follows, all of the procedures being described under “Experimental Procedures.” CVP8/1/P2(EcoRI res) DNA I was labeled with 32P by nick translation. The DNA was digested with R.HindIII at 37” for 6 h. Then an amount of enzyme equal to that originally used was added and incubation continued for 16 h more: the same amount of enzyme was added again and incubation was continued for 7 h more. The sample (0..2 ml) was extracted with phenol, the aqueous layer was passed through a column of Sephadex G-50, equilibrated with eleotrophore- sis buffer diluted lo-fold. The radioactive fractions (Cerenkov count- ing) were pooled, concentrated by lyophilization, and electrophoresed on a 1.4% agarose slab gel at room temperature for 4 h at 100 V. The bands of DNA fragments were detected by radioautography, the appropriate areas were cut out and homogenized in 1.5 mM NaCl, 0.15 mM sodium citrate and agarose fragments were removed by passing the material in each band through Sephadex G-25. Thereaf- ter the material was extracted with phenol and passed through Sephadex G-50. The counts per min in each band were determined and per cent of the total calculated. The molar ratios of the (HindID)- A, -B, and -C fragments were calculated as described in Table III.

Fragment

A B C

Size Total counts per min Molar ratio

% 9%

17 22 1.0 13 34 2.0 10 44 3.4

fragments (Hap ID-A and -B (Table II). Most, if not all of these sequences have been localized in (HindII/HindIII)-E (5, 15). Radioactively labeled cRNA transcripts of (HindII/HindIII)-E have been prepared and characterized (5). In the experiment shown in Fig. 9,5 a 3ZP-labeled cRNA of (HindII/HindIII)-E was used as a probe to localize the (HindIIIHindIID-E sequences in the fragments generated by endonuclease R.HindIII cleavage of 3H-labeled CVP8/1/P2(EcoRI res) DNA I. After digestion the products were electrophoresed on an agarose slab gel and the fragments were then transferred directly to a sheet of nitrocellulose. The 32P-labeled cRNA was hybridized to strips of the nitrocellulose, and the strips were cut into segments and counted. In Fig. 9, the 3H counts show the location of (HindHI)-A, -B, and -C. In Fig. 9a, the “P counts indicated that sequences homologous to (HindIIl HindIIIl-E are present in (HindIII)-C, the shortest of the three fragments. A small amount of hybridization of the cRNA in the region of (Hi&III)-A was also observed, although the peak of 32P counts does not coincide exactly with the peak of 3H. The reason for this small amount of hybridization is not known, but it may reflect less than total digestion. Fig. 9b is a control in which the 3ZP-labeled probe was total CVP8/1/P2(EcoRI res) DNA.

FIG. 8. Restriction endonuclease fragments of CVP8/1/P2(EcoRI res) DNA I. The various 32P-labeled fragments were digested and then electrophoresed on a polyacrylamide slab gel. A radioautogram of the gel is shown. The letters at the right correspond to the (HindII/ Hind1111 fragments of CVP8/1/P2(EcoRI res) DNA I. The letters at the left correspond to (HindIID-B and -C of CVPS/l/P2(EcoRI res) DNA I. a, Strain 777 SV40 DNA with R. (HindIIIHindIII), only the A fragment is clearly visible at the top; b, CVP8/1/P2(EcoRI res) (HindrID-B; c, as b; d, CVP8/1/P2(EcoRI res) (HindIID-B with R. (HindIIIHindIII); e, CVP8/1/P2 (EcoRI res) (HindIID-C fragment; f, as e; g, CVP8/1/P2(EcoRI res) (HindIID-C fragment with R. (HindIIIHindIID; some undigested (HindID)-C remains; h, CVP8/ l/P2(EcoRI res) DNA with R. (HindIIIHindIII); i, CVP8/1/P2(EcoRI res) DNA with R.HindII; j, as a.

Given the stoichiometry shown in Table IV, it seems reasonable to assume that the (Hap ID-A fragment may give rise to one copy each of (HindID)-A and one of (HindID)-C, while the (Hap 111-B fragment gives rise to one copy each of (HindID)-B and (HindUI)-C. According to this interpretation, (HindIID-A differs from (HindID)-B by the presence of (HindII/HindIII)-D and thus (HindII/HindIII)-D is characterized as a fragment generated by endonuclease R.HindII alone. Therefore, in Fig. 1, (HindID-D is placed between (HindII/HindIII)-A and -C in

(Hap ID-A. This interpretation is supported by the data in Slot i of Fig. 8, which shows a digestion of CVP8/1/P2(EcoRI res) DNA I with endonuclease R.HincII. Three main products ((HincID-A, -B, and -0 are seen (in addition to some partially digested material at the top of the gel), corresponding to 17, 7.7, and 4.5% of a full length SV40 genome, respectively. The 17% fragment should correspond to a combination of (HindIIl

HindID)-A and -B fragments, the 7.7% fragment to a combination of (HindII/HindIII)-C and -E, and the smallest fragment (HincII)-C, which migrates in the same manner as (HindIIl HindID)-D fragment (compare Slot i with Slot h in Fig. 8), would be that fragment itself, generated from the (Hap ID-A portion of the molecule.4

5 We are grateful to Dr. Shoshana Segal for her help in carrying out the experiments described in Table IV and Fig. 9.

Distribution and Characterization of SV40 and Monkey Sequences in CVPBIl IP2(EcoRI res) DNA - Earlier experiments (15) on the mixture of defective SV40 variants from which CVP8IlIP2(EcoRI res) was derived, suggested that the (HindII/HindIII)-A and -B fragments would contain wild type


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Structure ofa Host-substituted SV40 Variant 5131

10 20 30 40 50 al

STRIP NUMBER

FIG. 9. Digestion of CVP8IlIP2(EcoRI res) DNA I with endonuclease R.HindIII and hybridization of the fragments to 32P-labeled cRNA of (HindII/HindIII)-E. 3H-labeled CVP8/1/P2(EcoRI res) DNA I (3 fig) was digested with endonuclease R.HindIII and then electrophoresed, on a 1.4% agarose slab gel. The sample was applied as a streak across the width of the gel. After electrophoresis the gel was stained with ethidium bromide to ascertain proper band separation and the DNA fragments were then transferred to a sheet of nitrocellulose as described under “Experimental Procedures.” The sheet was cut into B-mm-wide lengthwise strips and strips were hybridized at 37” for 41 h with a, 32P-labeled cRNA of (HindII/HindIII)-E, 5000 cpm or b, 32P-labeled CVPBIlIP2(EcoRI res) DNA I prepared by nick translation as described under “Experimental Procedures.” After washing and air drying, the strips were cut into 2-mm pieces and 3H and 32P were counted after dissolving the nitrocellulose with methyl cellosolve (O-O), 3H-labeled CVPB/l/P2(EcoRI res) Hind111 fragments; (O- - -01, 3zP-labeled probes.

SV40 sequences, that (HindII/HindIII)-C fragment would hybridize little, if at all, to either SV40 or monkey DNA on filters, and that (HindII/HindIII)-E would contain no SV40 sequences detectable by filter hybridization but would hybridize efficiently to monkey sequences. As already described, (HindIIIHindIIIl-E fragment did indeed hybridize readily to filters containing monkey DNA (5, 15) indicating that it contains sequences derived from the repetitive Portion of the monkey genome. The data in Table V5 show the results obtained when 32P-labeled (HindII/HindIII)-A, -B, -C, -D, and -E fragments were hybridized to filters containing either monkey, wild type SV40, or total CVPS/l/P2 DNA. The latter filters serve as a positive control for this experiment since all the sequences in question are present in the DNA that is their source. This positive control is a measure of the efficiency of hybridization that can be expected in each instance. Thus, (HindII/HindIII1-A and -B hybridize as well or better to the filters containing SV40 DNA as to the filters containing the defective itself. (Note that the filters containing CVP8/1/P2 DNA used with (HindII/HindIII)-B fragment contained half as much DNA as those used in the other experiments). A small amount of hybridization over the blank was observed for both these fragments when the filters contained monkey DNA; the significance of these values is not known. Thus, (HindIIl HindIII)-A and -B contain SV40 sequences, although they may of course contain other sequences as well. (HindII/HindIID-C fragment hybridized very efficiently to filters containing the defective DNA, but little, if at all, to either wild type SV40 DNA or monkey DNA. Thus, (HindII/HindIII)-C fragment may contain either sequences homologous to the unique class of monkey sequences, or some sequence foreign to both SV40

TABLE V

Hybridization of CVP8IlIP2(EcoRI res) (HindIIlHindIII) fragments to SV40 and monkey DNA on filters

Hybridization was carried out as described under “Experimental Procedures.” The isolated fragments (HindIIIHindIIIl-A, -B, -C, and -E were labeled with 32P at the single 5’ terminus corresponding to cleavage by endonuclease R.HindIII: fragment D, which contains two ends generated by R’HindII cleavage was labeled at both 5’ termini as described under “Experimental Procedures.” A filter with no DNA was included in each incubation: the per cent of the input radioactivity found on the blank filters was uniformly low and the values reported (“no DNA” column) are averages of all blanks. The monkey DNA was isolated from BSC-1 monkey kidney cells and 17.5 pg of DNA was on each filter except for the experiment with fragment D, when 12.5 pg of DNA was on each filter. SV40 DNA was from strain 777 wild type plaque purified virus and 6 +g were on each filter except with fragment D, when 2.5 pg were present. Total CVP8/1/P2 DNA I was used and 12.5 +g of DNA were on each filter except for the experiments with fragment B and D when 6 and 5 &g were present, respectively.

Per cent hybridized to filter containing “P-labeled fragment Input

DNNOA

MOW key

DNA

sv40 DNA

CVPB/ l/P2

DNA

CPm

A 6010 0.1 1 20 23 B 4518 0.1 1 23 9 C 1821 0.6 2 7 80 D 10,285 1.7 4 6 11 E 1409 0.6 71 6 108

and monkey. (HindID-D fragment did not hybridize efficiently even to CVPWP2(EcoRI res) DNA, perhaps reflecting the very low concentration of (HindID-D sequences in the defective. The low per cent of hybridization to SV40 DNA or monkey DNA precludes firm conclusions concerning the nature of the sequences in (HindID-D. A separate experiment with one- fourth as much (HindID-D fragment and twice as much SV40 DNA on the filter also yielded 6% hybridization, suggesting that the concentration of SV40 DNA on the filter was not a limiting factor. The (HindII/HindIII)-E fragment gave the expected result (5, 15).

The experiments described in Table VI were designed to obtain evidence concerning which of the DNA sequences of wild type SV40 DNA are present in the defective genome. 3zP- labeled CVP8/1IP2(EcoRI res) DNA I was fragmented and denatured and then mixed with separated denatured 3H-labeled wild type SV40 (HindIIIHindIII) fragments. The mixed fragments were then subjected to another cycle of denaturation before being allowed to reanneal. At the end of a predeter- mined reannealing period, two samples were removed and one was treated with single strand specific Sl nuclease. The trichloroacetic acid insoluble counts were determined in each case and the per cent of the 3H and 32P counts that became resistant to the nuclease were determined. The per cent of the 3H-labeled wild type fragment that reannealed was then used to normalize the data on the annealing of the 32P-labeled defective. Because the number of 32P counts were low, two separate experiments are presented. The results suggest that about 15 to 20% of the sequences in the defective variant were derived from the wild type (HindII/HindIII)-A fragment; that about 10% may have been derived from the wild type (HindIIl HindUI)-B fragment; that about 20% may have been derived from either the wild type (HindII/HindIII)-C or -D fragment or both (the strain 777(HindII/HindIII)-C and -D fragments are


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


TABLE VI

Hybridization of 3H-labeled R (HindIIlHindIII) fragments of wild type SV40 DNA with xzPP-labeled CVP8Il lP2(EcoRI res) DNA I

Experiment 1: 32P-labeled CVP8/1/PZ(EcoRI res) DNA I was heated (at about 0.05 pg/ml) for 15 min at 100” in 1.5 ITIM NaCl, 0.15 rnM sodium citrate. It (0.08 ml) was then mixed in a final volume of 0.9 ml of the same buffer with individual 3H-labeled 777 SV40 DNA (HindIIIHindIII) fragments (approximately 0.05 pg), and 20 pg of sonicated, heat denatured Escherichia coli DNA. The mixture was then heated for 20 min at loo”, chilled rapidly in an ice salt bath, and 0.2 ml of 10 times concentrated, cold hybridization buffer was added. Two 0.2-ml samples were removed (zero time) and the rest of the mixture was incubated at 68” for 105 min at which time another two 0.2-ml samples were removed. One each of the samples from 0 and 105 min was treated with Sl nuclease as described under “Experi- mental Procedures”; the second sample was incubated under the same conditions without Sl nuclease and in each case the trichloroacetic acid precipitable counts per min were determined. The average of the precipitable counts per min obtained from samples without Sl were taken as the input radioactivity. The precipitable counts per min in the zero time samples treated with Sl were, for 32P, less than 2% of the total in each case, except as indicated. These values were not subtracted. The number in the column marked 3’P/3H is the ratio multiplied by 100 and this calculation normalizes the 32P hybridization to 100% reannealing of the wild type fragments. The filters were counted for 10 min. The input 3H was between 2000 and 5000 cpm; the input 32P was 130 cpm, in each 0.2-ml sample. Background was 15 cpm.

Experiment 2: Identical to Experiment 1 except as follows. The 32P-labeled CVP8/1/P2(EcoRI res) DNA I was initially heated at 0.01 pg/ml and the total 3H-labeled 777 SV40 DNA was heated in the same way at 0.2 pg/ml. The denaturation and reannealing of the mixture was done on twice the scale of Experiment 1 and the volume of each sample removed for Sl treatment was 0.4 ml. Annealing was for 2 h. The input 3H was between 4000 and 9000 cpm; the input 32P was 60 cpm, in each 0.4-ml sample.

3H-labeled Per cent counts per min resistant to Sl GYindIIl

HindIII) frag- merit of sv40

Experiment 1 Experiment 2

(777) 3H 32P 3ZP/3H 3H “P 32P/3H

A 71 11 15 71 14” 19 B 91 9 10 C+D 79 15 19 69 16 23 E 69 5 8 81 8 10

F 79 3 4.2 65 2 4 G 82 9 11 ?3 6 8 H 51 4 7.8 58 2 4 I 59 1 1.5 78 3 4 J 62 6 10.3 69 36 4 K 70 3 3.8 67 1 2 None 4 2 Total 771 38 22 57

a Sl resistant 32P counts per min zero time, 6%. b Sl resistant 32P counts per min at zero time, 4%.

not separable)6; that small portions of the wild type (HindIIl HindIII)-E and -G fragments may be present in the defective. The results with the other fragments indicate that none of the sequences of the other wild type (HindIIIHindIII) fragments were detectable by these experiments. Summation of the percentages of the defective sequences that are homologous to the various wild type (HiadIIIHindIII) fragments indicates that about 50% of the total sequences in the defective are derived from wild type SV40 sequences. As shown in the last line of Table VI, when randomly fragmented total 3H-labeled strain 777 wild type DNA was substituted for the separated frag-

B Alan Kay, unpublished experiments.

ments in the hybridization reaction, about 57% of the defective DNA appeared to be homologous, a number in good agreement with the sum obtained from hybridization with the separated fragments.

DISCUSSION

At the start of this work, our aim was to isolate a single defective DNA containing sequences derived from the monkey genome. Attempts to purify such a species from the mixture of defective genomes present in the CVPWP2 passage by a cloning procedure were not sucessful(2). A mixture containing (in addition to a small amount of wild type material) two classes of defective species, one of which did not contain sequences derived from monkey repetitive DNA (CVP8/1/ P2(EcoRI sens) DNA I) and one of which did (CVP8/1/ P2(EcoRI res) DNA I), was obtained (2). Treatment with endonuclease R.EcoRI converted wild type SV40 DNA I and CVPWl/P2(EcoRI sens) DNA I into DNA III, but the species containing monkey DNA was resistant to the nuclease and remained in the form of DNA I, thus permitting convenient purification of CVP8/1/P2(EcoRI res) DNA I (2). Physical characterization of CVP8/1/P2(EcoRI res) DNA I indicated that the preparation consisted of material that was slightly smaller than wild type SV40 DNA I, and close to homogeneous in size. Occasionally the data suggested that several species, very close in size and barely separable, might be present (2).

The data presented in this report support the view that CVP8IlIP2(EcoRI res) DNA is composed of 1, or a few very closely related molecules. For ease of discussion, the DNA I molecules can be described as four segments, each being one of the fragments generated by cleavage with endonuclease R Hap II. Two different fragments, (HindII)-A and -B, are generated by this cleavage and are approximately 26 and 23% of a wild type genome in length, respectively. The molar ratio of the two fragments is about 2 to 3 (Hap 111-B to 1 (Hap 11)-A. Thus it is possible that CVP8Il/P2(EcoRI res) DNA I is composed either of molecules containing three copies of (Hap 11)-B and one of (Hap 11)-A, or of a mixture of molecules each made up of different numbers of (Hap 111-B and (Hap 11)-A, but yielding the observed molar ratio as an average. The data do not permit us to distinguish between the many possibilities, but it does seem unlikely that molecules containing four (Hap 11)-A fragments are very abundant since CVP8/1IP2(EcoRI res) DNA I appears to be somewhat shorter than a full length wild type genome (2).

Fig. 10 shows a schematic drawing of 1 possible molecule composed of four copies of the (Hap 111-B fragment. The struc- tures of (Hap 11)-A and (Hap II&B, as determined by restriction endonuclease cleavage, are summarized in Fig. 1. All of the available data are consistent with the notion that the two fragments are identical, except for the presence of (HindIIl HindIII)-D fragment (or the equivalent, (HincII)-C) in (Hap WA. The presence of a R.Hup II cleavage site in the (HindIIl HindIII)-B fragments of both (Hap 11)-A and (Hap ID-B is indicated by the absence of (HindII/HindIII)-B after cleavage of both separated Hap II fragments with endonuclease R. (HindIIIHindIII) (Fig. 6). Similarly, the disappearance of (HindII/HindIII)-A after cleavage with R.&m I (Fig. 3) as well as the formation of fragments of similar size after treatment of either (Hap ID-A or (Hap 11)-B with endonuclease R.Bum I support the relatedness of (Hap ID-A and (Hap II&B. While analysis of the (HindIUHindIII) fragments generated by cleavage of (Hap 11)-A and (Hap 111-B do not unequivocally cor&-m the presence of (HindII/HindIII)-C and -E fragments


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


FIG. 10. A schematic drawing of one possible species present in CVPWl/P2(EcoRI res) DNA I. namely one composed of four reitera- tions of the (Hap ID-B fragment. The-fragments generated by cleavage with endonuclease R. (HindIWfindIII) are indicated on the inner circle. On the inner circle, an unbroken line indicates possible SV40 sequences; the jagged line, sequences derived from the repetitive class of monkey DNA; the small circles, sequences which are neither SV40 nor repetitive monkey sequences.

in each because of the fortuitous identity in size of (HindII/ HindIII)-C and C’ on the one hand, and (HindII/HindIII)-E and -E’ on the other, both the stoichiometry of the (HindIIl Hind1111 fragments and the hybridization of both (Hap ID-A and (Hap 11)-B to monkey DNA on filters (Table II) indicate that (HindII/HindIII)-C and -E are present in both (Hap 111-A and (Hap ID-B. In addition, nucleotide sequence analysis indicates that both (HindII/HindIII)-C and -E, isolated from intact total CVP8/1/P2(EcoRI res) DNA I are single, homogeneous sequences,3 thus supporting the view that (Hap 111-A and (Hap ID-B are likely to be identical, except for the presence of the (HincII)-D fragment.

The analysis with restriction endonucleases permits the positioning of the various (HindIIIHindIII) fragments with respect to one another, as shown in Fig. 1. The hybridization data indicate that (HindII/HindIII)-E contains primarily DNA sequences derived from the monkey genome since it hybridized little, if at all, to SV40 DNA. The hybridization data also indicate that (HindII/HindIII)-C contains few if any SV40 DNA sequences, nor does it appear to contain sequences derived from the repetitive class of monkey DNA. It is of course possible that some short stretch of sequences derived from SV40 is present in both (HindII/HindIII)-C and -E but not detectable by the hybridization. It seems probable that the sequences in (HindII/HindIII)-C fragment were derived from monkey sequences of low reiteration frequencies, or from unique monkey sequences as the presence of such sequences was indicated in a mixture of defectives that was a parent of CVP8/1/P2(EcoRI res) DNA I (24, 25) as well as in similar but independently derived defectives (3, 4). The sequences in (HindII/HindIII)-C may nevertheless represent sequences un- related either to SV40 or monkey DNA, and work on the characterization of (HindII/HindIII)-C fragment is continuing in this laboratory. The data presented here demonstrate that (HindIIIHindIID-C and -E are contiguous within CVP8/1/ P2(EcoRI res) DNA I and are separated from one another by a R. Hind111 cleavage. Together, (HindII/HindIII)-C and -E comprise approximately 7.8% of a full length genome. Consid-

ering that they each occur four times in the molecules and that some molecules may be only about 92% of the wild type genome in length, between 31 and 34% of the molecule may be composed of sequences derived from the monkey genome. The data in Table VI indicate that about 50 to 60% of the total sequences are SV40 sequences and therefore that approximately 40 to 50% of the molecule may contain monkey sequences. Thus it is possible that the (HindIIIHindIID-A, or-B fragments, or the (HincII)-D fragment also contain monkey sequences.

Only preliminary data on the nature of the SV40 sequences that are conserved in CVP8/1/P2(EcoRI res) DNA I are presented here. The data (Table VI) suggest that segments associ- ated with wild type (HindII/HindIII)-A, and -C, and/or -D are present. There is also an indication that sequences occurring in wild type (HindII/HindIII)-B, -G, and -E may be present but the data do not permit firm conclusions. It has been demonstrated previously by several groups of investigators that those defective genomes with sufficient selective advan- tage to accumulate in substantial amounts during passaging contain those SV40 sequences corresponding to the site of initi- ation of replication on SV40 (3, 4, 26-29). This site is contained within the wild type (HindII/HindIII)-C fragment (30, 31). Therefore it is reasonable to assume that segments of wild type (HindII/HindIII)-C fragment are present in CVP8/1/ P2(EcoRI res) DNA I. The single site of cleavage of endonuclease R.Hup II in wild type SV40 DNA is within the (HindIIl HindIII)-C fragment (8, 22). It is thus possible that the R.Hup II site in (HindII/HindIII)-B of CVP8IlIP2(EcoRI res) DNA I corresponds to the wild type site. Similarly, the single site of cleavage of endonuclease R.Bum I in wild type SV40 is within the (HindII/HindIII)-G fragment. The endonuclease R.Bam I site in the (HindII/HindIII)-A fragment of CVP8IlIP2(EcoRI res) DNA I may correspond to the wild type R-Bum I site. It is clear from the data in Table VI that only a portion of each of the detectable wild type (HindIIIHindIII) fragments can be present in CVP8/1/P2(EcoRI res) DNA I. For example, 15 to 19% of CVP8Il/P2(EcoRI res) DNA I hybridizes to wild type (HirzdII/HindIII)-A fragment; assuming four repeats of all sequences in the defective variant, the homologous defective segment would be only 4 to 5% of a wild type genome in length. Since wild type (HindII/HindIII)-A is 22% of a genome long (lo), the defective variant can contain only about 20% of the wild type (HindII/HindIII)-A sequence.

In recent years the defective variants of SV40 that arise upon serial passage of wild type, plaque purified virus in monkey kidney cells in tissue culture have been studied exten- sively. The DNA molecules of several such variants have been isolated, purified, and characterized. Although the diverse DNAs have not been compared in detail as yet, it appears that they are not identical. Nevertheless there are certain features of these genomes which occur frequently, and the molecule described in this report, CVPB/l/P2(EcoRI res) DNA I, shares those common features. All the variant DNAs are double- stranded, closed circular duplex superhelical molecules (2-4, 13-15, 26, 28, 29, 32-38) as is the wild type genome. They are, typically, between 70 and 100% of a wild type genome in length; this size range appears to define the requirement for encapsidation (3, 29). Many of the variant DNAs contain tandem repetitions of portions of the wild type genome (called “reiteration mutants”) and thus also lack other wild type sequences (3, 4, 28, 29, 33, 34). Each of the repeated segments in the reiteration mutants contain those SV40 sequences that correspond to the origin of viral DNA replication (3, 4, 27, 28)


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


and the defectives depend on functions supplied by helper genomes for replication (29). Many of the variants contain DNA sequences derived from the monkey genome (called “sub- stitution mutants”) (2, 3, 4, 7, 13-15, 29, 34). Those substitu- tion mutants that have been characterized are also reiteration mutants and consist of tandem repeats of DNA segments containing both monkey and SV40 sequences (3, 4)‘; the monkey sequences may be derived from either the unique or repetitive class of DNA, or both (3, 4, 13-15, 24, 25, 29). The

CVP8IlIP2(EcoRI res) DNA I then falls in the class of substituted-reiteration mutants: it is a double-stranded, closed circular superhelical DNA, a few per cent shorter than a full length wild type genome (2); it contains repetitive monkey DNA sequences and very likely unique monkey DNA sequences as well; and it is made up of four tandem repeats of DNA segments containing both monkey and SV40 sequences. The hybridization data are consistent with the possibility that CVP8/ l/P2(EcoRI res) DNA I contains the sequences corresponding to the origin of replication within the wild type (HindIIl HindIID-C fragment. It is of interest to point out that other wild type sequences detected in CVP8/1/P2(EcoRI res) DNA I may also be common to several separately isolated substituted-reiteration mutants, namely, sequences found in wild type (HindII/HindIII)-A fragment (3, 4), in wild type (HindIIl HindIID-G fragment (4) and perhaps at least a small segment of wild type (HindII/HindIII)-B fragment (4).

There are a variety of interesting questions raised by the structure of the many defective variants of SV40 that have been studied. One of these questions centers on the nature of the monkey sequences that are incorporated into substituted defectives. There is substantial evidence indicating that these

sequences represent a nonrandom portion of the monkey genome (24, 25).3 It is perhaps not surprising that highly reiterated monkey sequences appear frequently in the substituted defectives. However, the occurrence of the same monkey sequences of low reiteration frequency in independently isolated substituted defectives (25) suggests a preferred region for recombination between SV40 DNA and the DNA of the permissive monkey cells. Another interesting opportunity provided by the substituted defective variants of SV40 is the study of the nucleotide sequences at the sites of recombination between SV40 DNA and monkey DNA.

The experiments reported here indicate that two segments of DNA, containing few, if any, SV40 sequences and either highly reiterated ((HindII/HindIII)-E) or what is likely to be infrequently reiterated or unique monkey DNA ((HindII/ HindIII)-C) can readily be obtained from CVP8/1/P2(EcoRI res) DNA I. The availability of these segments has permitted investigation of the organization of the sequences within the monkey genome (5)” as well as the nucleotide sequence of portions of the monkey genome.3

Acknowledgments-We are grateful to Drs. R. DiLauro, M. Botchan, M. Rosenberg, and S. Segal for helpful discussions and assistance. We also thank Drs. S. Segal, E. Kuff, C. Klee, and M. Rosenberg for their critical reading of the manuscript.

REFERENCES 1. Smith, H. O., and Nathans, D. (197315. Mol. Biol. 81,419-423

2.

3.

4.

5.

6.

7.

8. 9.

10.

11.

12.

13. 14.

15.

16. 17.

18.

19.

20. 21. 22.

23.

24. 25.

26. 27.

28.

Rao, G. R. K., and Singer, M. F. (1977) J. Biol. Chem. 252, 5115- 5123

Lee, T. N. H., Brockman, W. W., and Nathans, D. (1975) Virol- ogy 66, 53-69

Davoli, D., Ganem, D., Nussbaum, A. L., Fareed, G. C., How- ley, P. M., Khoury, G., and Martin, M. A. (1977) Virology, 77, 110-124

Segal, S., Garner, M., Singer, M., and Rosenberg, M. (1976) Cell 9, 247-257

Takanami, M. (1974) in Methods in MolecularBiology (Wickner, R., ed) Vol. 7 pp. 114-125, Marcel Dekker, Inc., New York

Sharp, P. A., Sugden, B., and Sambrook, J. (1973) Biochemistry 12, 3055-3063

Wilson, G. A., and Young, F. E. (1975)J. Mol. Biol. 97, 123-125 Smith, H. 0. (1974) in Methods in Molecular Biology, (Wickner,

R. B., ed) Vol. 7, pp. 71-85, M. Dekker, Inc., New York Danna, K. J., Sack, G. H., Jr., and Nathans, D. (1973) J. Mo2.

Biol. 78, 363-376 Old, R. W., Murray, K., and Roizes, G. (1975) J. Mol. Biol. 92,

331-339 Landy, A., Ruedisueli, E., Robinson, L., Foeller, C., and Ross,

W. (1974) Biochemists 13. 2134 Lavi, S., and Winocour,“E. (1972) J. Vtiol. 9, 309-316 Lavi, S., Rozenblatt, S., Singer. M. F., and Winocour E. (1973) J.

- Viral. 12, 492-500 Rozenblatt, S., Lavi, S., Singer, M. F., and Winocour, E. (1973)

J. Viral. 12, 501-510 Vogt, V. M. (1973) Eur. J. Biochem. 33, 192-200 Kelly, R. B., Cozzarelli, N. R., Deutscher, M. P., Lehman, I. R.,

and Kornberg, A. (1970) J. Biol. Chem. 245, 39-45 Rigby, P. W. J., Rhodes, D., Dieckmann, M., and Berg, P. (1977)

J. Mol. Biol., in press Maniatis, T., Jeffrey, A., and Kleid, D. G. (1975) Proc. N&Z.

Acad. Sci. U. S. A. 72. 1184-1188 Southern, E. M. (1975)j. Mol. Biol. 98, 503-517 Botchan. M.. TOPP, W.. and Sambrook. J. (1976) Cell 9. 269-287 Fiers, W., Dan&, K., Rogiers, R., Vandevoorde, A., Van Her-

reweghe, J., Van Heuverswyn, H., Volckaert, G., and Yang, R. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 179-186

Abrahams, P. J., Mulder, C., Van De Voorde, A., Warnaar, S. 0.. and van der Eb. A. J. (1975) J. Viral. 16. 818-823

Frenkel, N., Lavi, S.,‘and Winocour, E. (1974) Virology 60, 9-20 Gren, M., Kuff, E. L., and Winocour, E. (1976) Virology 73, 419- -_

430 Mertz, J. E., and Berg, P. (1974) Virology 62, 112-124 Frenkel, N., Rozenblatt, S., and Winocour, E. (1975) in Tumor

Virus-Host CellInteraction (Kolben, A., ed) pp. 39-58, Plenum Publishing Corp., New York

Khoury, G., Fareed, G. C., Berry, K., Martin, M. A., Lee, T. N. H. and Nathans, D. (1974) J. Mol. Biol. 87, 289-301

29. Ganem, D., Nussbaum, A. L., Davoli, D., and Fareed, G. C. (1976) J. Mol. Biol. 101, 57-83

30. Danna, K. J., and Nathans, D. (1972) Proc. N&l. Acad. Sci. U. S. A. 69, 3097-3100

31. Fareed, G. C., Garon, C. F., and Salzman, N. P. (1972) J. Viral. 10, 484-491

32. Yoshiike, K. (1968) Virology 34, 391-401 33. Tai, H. T., Smith, C. A., Sharp, P. A., and Vinograd, J. (1972) J.

Viral. 9, 317-325 34. Martin, M. A., Gelb, L. D., Fareed, G. C., and Milstein, J. B.

(1973) J. Viral. 12, 748-757 35. Fareed, G. C., Byrne, J. C., and Martin, M. A. (1974) J. Mol.

Biol. 87, 275-288 36. Brockman, W. W., Gutai, M. W., and Nathans, D. (1975) Virol-

ogy 66, 36-52 37. Brockman, W. W., Lee, T. N. H., and Nathans, D. (1973) Virol-

ogy 54, 384-397 38. Brockman, W. W., and Nathans, D. (1974) Proc. N&Z. Acad. Sci.

U. S. A. 71, 942-946


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

G R Rao and M F Singersequences derived from monkey. II. Structure of DNA.

Studies on a defective variant of simian virus 40 that is substituted with DNA

1977, 252:5124-5134.J. Biol. Chem.

http://www.jbc.org/content/252/14/5124Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/252/14/5124.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/content/252/14/5124

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;252/14/5124&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/252/14/5124

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=252/14/5124&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/252/14/5124

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/252/14/5124.full.html#ref-list-1

http://www.jbc.org/

Studies on a Defective Variant of Simian Virus 40 That Is Substituted ...

Documents

Transcript of Studies on a Defective Variant of Simian Virus 40 That Is Substituted ...